Method for the diagnosis of and/or monitoring mucormycosis

ABSTRACT

A method is described for the diagnosis and/or monitoring of active or previous infection by Mucor which consists in the identification of Mucorales-specific T cells in samples from biological fluids taken from the patient and put into contact with a Mucor antigen. These specific immune responses can be detected by the execution of immunoenzymatic assays (ELISPOT, Quantiferon) or of immunocytofluorimetric assays [Cytokine Secretion Assay (CSA), Intracellular Cytokine Staining (ICS)] in vitro. In greater detail, the method in question provides for checking for the presence of specific IFN-γ producing T cells, of specific IL-10 producing T cells and/or specific IL-4 producing T cells.

The present invention relates to a method for the diagnosis and/ormonitoring of active or previous infection by Mucor which consists inthe identification of Mucorales-specific T cells in samples ofbiological fluids which have been taken from the patient and put incontact with a Mucor antigen. These specific immune responses can bedetected by performing immunoenzymatic assays (ELISPOT, Quantiferon)(Lalvani, Chest., 131: 1898-1906, 2007) or immunocytofluorimetric assays[Cytokine Secretion Assay (CSA) (Goodyear et al., Blood, 112: 3362-3372,2008)], Intracellular Cytokine Staining (ICS) (Jaimes et al., J.Immunol. Method, 1-15, 2010)] in vitro, which are incorporated herein byreference. In greater detail, the method in question provides forchecking for the presence of specific IFN-γ producing T cells, ofspecific IL-10 producing T cells, and/or of specific IL-4 producing Tcells.

STATE OF THE ART

Invasive fungal infections (IFIs) represent an important cause ofmorbidity and mortality in patients suffering from malignant haemopathywho undergo not only allogeneic bone marrow transplantation but alsostandard chemotherapy. The risk of death in patients suffering from IFIsreaches percentages as high as 90%.

The aetiological agent that is mostly responsible for IFIs isrepresented by fungi of the species Aspergillus which accounts forslightly more than 50% of all invasive fungal infections. However, inrecent years, fungi of the order Mucorales [Note by the inventors: Arecent reclassification abolished the order Zygomycetes and includedfungi of the order Mucorales in the provisional subphylumMucoromycotina, and fungi of the order Entomophthorales in that ofEntomophthoromycotina; pending a definitive reclassification and areintroduction of the order Zygomycetes, since the Entomophthoralescause low level and chronic infections that are almost never invasiveand never in patients with malignant haemopathies, in this text,invasive infections by Mucorales will be treated as synonymous with theinfections previously known as Invasive Zygomycosis] have become thesecond most common agent responsible for invasive fungal infections withpercentages variable from 3% to 15%, according to case histories.Invasive Mucorales (IM) infections have very high mortality percentagesof from 70% to 90% of patients affected. The causes of such highmortality lie in the lack of diagnostic methods that permit certain andtimely diagnosis of mucormycosis. It has been estimated that, in thecase of IM, a delay of six days in starting an effective antifungaltreatment after the appearance of the first symptoms of IM is associatedwith a doubling in mortality and a reduction in survival to 12 weeks forall but 20% of patients (Chamilos et al., Clin. Infect. Dis.; 47:503-509, 2008).

The diagnostic methods that are currently in use for the diagnosis of IMare represented almost exclusively by histological examination andcultural examination. However, both are greatly limited by: a)invasiveness and difficulty in obtaining a biopsy sample because of theclinical conditions of the patients affected; b) low sensitivity(cultural examination is positive in a percentage of about 10-20% ofcases (Lass-Florl. Clin. Microbiol. Infect.; 15: 60-65, 2009), c)contamination of the sample by environmental fungi (in one study only7.6% of cultural samples on which Mucorales were detected came frompatients actually affected by IM) (Torres-Narbona et al., J. Clin.Microbiol; 2051-2053, 2007); d) factors adversely affecting the growthof these fungi in culture, such as cold and protein inhibitors; e)difficulty in isolating Mucorales from cultural examinations ofperipheral blood (Chayakuleeree M, Eur. J. Microbiol. Infect. Dis.; 25:215-229, 2006).

Moreover, the radiological signs which are encountered most frequentlyin IM cases upon computerized axial tomography (CT), are not able todifferentiate IM from Invasive Aspergillosis since both pathogens areangioinvasive and often present the same radiological characteristics(Roden et al., Clin. Infect. Dis. 41: 634-653, 2005). Finally, thepolymerase chain reaction (PCR) for the amplification and theexamination of Mucorales nucleic acids on biological materials lacksstandardization and is used almost exclusively as a confirmation test onbiopsy material and not as a screening test on plasma and hence has thesame limitations of access as biopsy examination (Bialek et al., J.Clin. Pathol., 58: 1180-1184, 2005; Kasai et al, J. Clin. Micriobiol.,Vol. 46, No. 11, 3690-3702, 2008; Hata et al., J. Clin, Microbiol., Vol.46, No. 7, 2353-2358, 2008).

The diagnosis of IM is thus based on greater or lesser degrees ofprobability and is very rarely made with certainty, and often belatedlyin relation to the course of the infection. Recent studies, firstly inmice and subsequently also in man, have shown that adaptive immunity,that is, that represented by the T cells, plays a predominant role inthe host's defences against fungi. In particular, a cell-mediatedimmunity polarized to type 1 helper T cells (Th1) producing interferongamma (IFN-γ) is protective with respect to fungal infections,particularly Invasive Aspergillosis whereas a cell-mediated immunitypolarized to type 2 helper T cells (Th2) producing interleukin 10(IL-10) is permissive with respect to IA (Cenci et al. Infection andImmunity, Vol. 65. No. 2, 564-570, 1997; Hebart et al., Blood, Vol. 100,No. 13, 4521-4528, 2002; Romani, Nature Reviews, Vol, 4, 1-13, 2004).The ELISPOT method (Enzyme-linked ImmunoSpot Assay) has been shown to bea sensitive and specific tool in the diagnosis of infection bytuberculosis mycobacteria or in infections of viral origin, both inimmunocompetent patients and in immunosuppressed patients, by means of asearch for T cells that are specific to the mycobacterium (Lalvani, Am.J. Respir. Crit. Care Med., Vol. 166, 789-790, 2002) or by the isolationof T cells that are specific to the Epstein-Barr virus in patientsundergoing kidney transplant (Comoli et al., Blood, 99: 2592-2598,2002).

ELISPOT is an assay based on the cell immunological response to aspecific antigen. This method is generally implemented on plates with 96wells, coated with specific monoclonal antibodies, in which the cellsand the specific antigen are incubated for 6-48 hours.

During this period, the cell response takes place and the specificcytokines are produced, according to the type of antibody/antigen used,and give rise to spots that are detected and counted by conventionalimage-analysis software.

The ELISPOT method for a generic evaluation of blood cell activity isdescribed in European patent application EP0957359 (Volkmar et al.),which is incorporated herein by reference.

The ELISPOT method applied to T lymphocytes for the diagnosis ormonitoring of diseases such as hepatitis B, hepatitis C, tuberculosis,malaria, HIV or influenza is described in International patentapplication WO98/23960 (Lalvani et al.), which is incorporated herein byreference.

The ELISPOT method applied to T lymphocytes that are reactive toantigens derived from the protein ESAT-6, which is expressed byMycobacterium tuberculosis, for the diagnosis of tuberculosis, or evenpurely contact of the host with the pathogen (latent disease) isdescribed in International patent application WO00/26248 (Lalvani etal.), which is incorporated herein by reference.

A further ELISPOT method applied to T lymphocytes that are reactive notonly to antigens derived from ESAT-6 but also from the protein CFP-10 isdescribed in International patent application WO2004/005925 (Lalvani etal.), which is incorporated herein by reference.

The ELISPOT method applied to T lymphocytes which are reactive toantigens of hyphae and conidia of Aspergillus fumigatus for thediagnosis of Invasive Aspergillosis is described in International patentapplication WO2008/075395 (University of Modena and Reggio Emilia),which is incorporated herein by reference.

Up to now, the ELISPOT method has thus been used for diseases of viralorigin and for bacterial infections, in particular for infections byMycobacterium tuberculosis whereas, for fungal infections, it has beenused solely for the invasive form of infections by Aspergillusfumigatus.

DESCRIPTION OF THE INVENTION

It is known that immunoenzymatic assays, in particular ELISPOT assays,and immunocytofluorimetric assays can advantageously be used fordiagnosis, in particular early diagnosis and/or monitoring also offungal infections, particularly in the case of Invasive Mucormycosis(IM).

The objective of the invention is to utilize these assays and, inparticular, the ELISPOT assay, for the identification and counting of Tcells specific to Mucorales, so that simply the detection of IFN-γproducing cells, of IL-10 producing cells and/or of IL-4 producing cellscan be used for the determination of the risk of infection, for thediagnosis of IM, for the monitoring of active or previous infection, andfor improving the clinical management of patients who are at risk ofand/or suffering from this pathology.

The subject of the present invention is therefore a method for thediagnosis and/or monitoring of IM which comprises the execution of animmunoenzymatic or immunocytofluorimetric assay in vitro onmicrotitration plates such as, for example, the type with 96 wells,characterized in that the biological fluid taken from the patient is putinto contact with an antigen of fungi of the Mucorales class, inparticular an extractable Mucorales antigen.

According to one aspect of the present invention, the method permits theidentification and counting of T cells that are specific to Mucorales inorder to check for the presence of and/or to define a ratio betweenthose producing IFN-γ, those producing IL-10, and/or those producingIL-4.

According to the present invention, the immunoenzymatic assay is ELISPOTassay (Enzyme-Linked ImmunoSpot Assay) and the antigen, which ispreferably an extractable antigen, can be obtained from conidia ofMucorales or from hyphae of Mucorales.

Conidia (or conidiospores) are the predominantly unicellularreproductive structures of the fungus which are formed at the tips ofthe hyphae which in turn are cylindrical unicellular or multicellularfilaments; when arranged on top of one another, these form the myceliumor the vegetative bodies of the fungi.

In particular, according to an embodiment of the present invention, theantigen is obtained from conidia of Mucorales which are collected afterculture for two-four days, preferably for three days, preferably onSaboraud agar medium, filtered through sterile gauze. After filtration,the conidia are placed in liquid Saboraud medium and stirred for about6-18 hours, preferably 12 hours, to achieve germination which enablesthe antigenic determinants to be displayed. The germinated conidia arewashed in saline solution (PBS), deactivated by heat, preferably forabout 1 hour at 100° C., and counted. The antigens are then treated bysonication, preferably with the use of an ultrasound homogenizerreaching a maximum power of 400 W and working at a frequency of 24 Khz.The conidia are subjected to 4 sonication cycles of about 10 secondsduration, each at about 70% of maximum power. Finally, the germinatedand sonicated conidia are stored in PBS or culture medium at betweenabout −30 and −10° c., preferably at −20° C.

According to another embodiment of the present invention, the antigen,which is preferably an extractable antigen, is obtained from hyphae ofMucorales which are homogenized, for example, with the use of a mortarwith beads made of an inert material, preferably glass beads in buffer,preferably 50 mM Tris-HCl buffer with a pH of about 7.5. Theproteinaceous extract thus obtained is recovered after centrifuging atbetween 10 and 14,000 rpm for about 10 minutes and kept at a temperatureof between about −30 and −10° C., preferably −20° C.

For the purposes of the present invention, the expression “Mucoralesantigen” thus means a constituent of the fungus structure which istreated in a manner such that it is capable of stimulating a specific Tcell response.

The biological fluid that is used according to the method of the presentinvention may be blood, bronchioalveolar lavage liquid (BAL), or pleuralliquid from patients who are at risk of or already infected byMucormycosis.

In particular, the fluid may be obtained by citrated endovenoussampling, preferably of about 20 ml, by citrated pleural liquidsampling, preferably of about 20 ml, or by citrated BAL liquid sampling,preferably of about 30 ml.

According to the method of the present invention, the mononucleatedcells or purely the isolated T lymphocytes from the endovenous bloodsamples may be used.

According to the present invention, the sample of blood, pleural liquid,or bronchioalveolar lavage liquid is diluted with an approximately equalvolume of culture medium, for example RPMI 1640, and is then centrifugedon density gradient (Ficoll, Lymphoprep) to separate the population ofmononucleated cells (PBMCs). After 2 washings, for example, in RPMI1640, the cells are frozen, preferably in liquid nitrogen, even morepreferably in the presence of 20% DMSO and 50% foetal bovine serum (FBS)for the ELISPOT tests.

The cell samples are then thawed slowly in a bath kept thermostaticallyat 35-40° C., preferably about 37° C. DNAse is added to a finalconcentration of about 10 mg/ml and left to act at ambient temperaturefor about 20 minutes. The cells are transferred into a test tube andresuspended carefully with about 10 ml of RPMI 1640 medium supplementedwith about 5% of FBS, which is added dropwise. After a firstcentrifuging at about 1,000 rpm for about 10′, the cell pellet obtainedis resuspended in R10 culture medium (RPMI 1640; 10% FBS; 1% sodiumpyruvate; 1% ampicillin; 0.5% gentamicin; 18 UI/ml IL-2) and the cellsare then counted in a Bürker chamber.

A “Pan Cell Isolation Kit, human” distributed by Miltenyi Biotech S.r.l.(Calderara di Reno, Bologna, Italy) has been used for the separation ofthe CD3+ T cells. The CD3+ T cells are separated by depletion of thenon-T cells (negative selection). The non-T cells, marked with acocktail of biotin-conjugated monoclonal antibodies (CD14, 16, 19, 36,56, 123 and Glycophorin A) are reacted with conjugated anti-biotinmonoclonal antibodies with magnetic micro-beads. The marked cells (non-Tcells) and the non-marked (CD3+ T cells) are passed through a MACS-MScolumn (Miltenyi) in the presence of a magnetic field, allowing the CD3+T cells to pass through and the non-T cells to be blocked. Theseparation process is carried out on cell samples after thawing,following the instructions provided by Miltenyi.

According to another embodiment of the present invention, the antigenmay be obtained from conidia of Mucorales which are collected afterculture for 2-4 days (for example, on Saboraud agar medium) by washingof the surface layer of the fungal culture with sterile water. Thewashing liquid obtained is filtered through sterile gauze andcentrifuged at between 10,000 and 15,000 rpm, preferably at about 12,000rpm, for 5-15 minutes, preferably 10 minutes. The supernatant liquid isremoved and the conidia pellet thus obtained is resuspended in liquidSaboraud and stirred for 6-18 hours, preferably for about 12 hours toachieve germination. The germinated conidia are washed in salinesolution (PBS), deactivated by heat, preferably for about 1 hour at 100°C., and counted. The antigen (2-4 million germinated conidia) is thentreated by sonication, preferably with the use of an ultrasoundhomogenizer reaching a maximum power of 400 W and working at a frequencyof 24 KHz. The conidia are subjected to 4 sonication cycles, each ofabout 10 seconds duration, preferably at 70% of maximum power. Finally,the germinated and sonicated conidia are resuspended in PBS or culturemedium (1 ml) and are used immediately or stored at about −20° C. andused subsequently. The conidia thus obtained are used in the ELISPOTtest at a final concentration of about 100,000-200,000 conidia/ml and inthe CSA test at a concentration of 200,000-400,000 conidia/ml.

According to a further embodiment of the present invention, theextractable antigen may be obtained from hyphae of Mucorales which arecollected by scraping of several culture plates with a scalpel blade andresuspended in sterile water. After vortexing, filtration is performedthrough sterile gauze to eliminate any agar. The mycete thus obtained iscentrifuged at about 15,000 rpm for about 10 minutes and then, when thesupernatant fluid has been removed, is frozen in liquid nitrogen forabout 12 hours. The thawed mycete is then homogenized in a mortar in thepresence of a volume of beads made of inert material, preferably glassbeads, which is equal to the volume of the mycete, in buffer, preferably50 mM Tris-HCl buffer at a pH of about 7.5. The proteinaceous extractthus obtained is recovered after centrifuging at about 14,000 rpm forabout 20′. After evaluation of the protein content, the hyphae extractis used in the ELISPOT test at a final concentration of about 6-10microgrammes/ml and in the CSA test at a final concentration of 10microgrammes/ml.

The antigen obtained may be used immediately or may be stored byfreezing to about −20° C. and used subsequently.

For the purposes of the present invention, the positivity threshold,that is, the minimum number of specific IFN-γ producing T cells,specific IL-10 producing T cells and/or specific IL-4 producing T cellsthat is considered indicative of the presence of Invasive Mucormycosisis between 2 and 10 SFCs, preferably 5 SFCs.

EXPERIMENTAL SECTION ELISPOT Method for Mucorales-Specific IFN-GammaProducing T Cells

96-well microtitration plates (Multiscreen HTS IP Sterile Plate),distributed by Millipore (Bedford, Mass., USA) and included in theELISPOT IFN-gamma kit distributed by Mabtech (Nacka Strand, Sweden) wereused for all of the ELISPOT tests. The bases of the wells of the plateswere made of nitrocellulose and were coated with a specific IFN-γmonoclonal antibody. The plates were removed from the packaging andwashed 4 times with PBS 1× (200 μl/well). The plates were fixed with R10(200 μl/well) and incubated at ambient temperature for ≧30′.

The fixing means were removed and 100,000-200,000 cells in R10 weredispensed into the individual wells (final volume 100-150 μl/well) andthen stimulated with sonicated, germinated conidia, deactivated by heat(1-2×10⁵ conidia/ml) or with proteinaceous extract (6-10 μg/ml) duringincubation at 37° C. in the presence of CO, (5%) for about 20 hours.

The positive controls were constituted by the same cells incubated withphytohaemagglutinin (PHA) (25 μg/ml) or with a human anti-CD3 antibody(Mabtech) used at a dilution of 1:1,000; the negative control wasconstituted by the cells alone without any stimulation. All of the testswere carried out in triplicate. After incubation, the plate was emptiedand subjected to 5 successive washings with PBS, 200 μl per well. Thecytokine-antibody complexes were stained by enzymatic reaction and theaddition of a chromogenic substrate: 100 μl of secondary antibodyconjugated with alkaline phosphatase, distributed by Mabtech, diluted1:200 in PBS 1× and 0.5% of foetal bovine serum (FBS) was dispensed intoeach individual well and, after incubation for 120′ at ambienttemperature, the substrate, 100 μl (BCIP/NTB-plus), also distributed byMabtech, was added. The reaction with the substrate was stopped after12-15′ by washing the plate with running water. The nitrocellulosemembrane was dried in air for at least 4 hours. Each individual spotwhich appeared on the base of the well corresponded to the secretion ofthe cytokine of a single cell (spot forming cell: SFC). The spots werethen counted automatically by means of an image-analysis tool (AIDELISPOT Reader System, Amplimedical) controlled by software capable ofproviding a quick and easy evaluation of the spots on the basis of theirsize and intensity.

ELISPOT Method for Mucorales-Specific IL-10 Producing T Cells

96-well microtitration plates (Multiscreen HTS IP Sterile Plate),distributed by Millipore (Bedford, Mass., USA) and included in theELISPOT IL-10 kit distributed by Mabtech (Nacka Strand, Sweden), wereused for all of the ELISPOT tests. The bases of the wells of the platewere made of nitrocellulose and were coated with a specific IL-10monoclonal antibody. The plates were removed from the packaging andwashed 4 times with PBS 1× (200 μl/well). The plates were fixed with R10(200 μl/well) and incubated at ambient temperature for ≧30′.

The fixing means were removed and 30,000-100,000 cells in R10 weredispensed into the individual wells (final volume of 100-150 μl/well)and then stimulated with sonicated, germinated conidia, de-activated byheat (1-2×10′ conidia/ml) or with proteinaceous extract (6-10 μg/ml)during incubation at 37° C. in the presence of CO₂, (5%) for about 40hours. The positive controls were constituted by the same cellsincubated with phytohaemagglutinin (PHA) (25 μg/ml) or with a humananti-CD3 antibody (Mabtech) used at a dilution of 1:1,000; the negativecontrol was constituted by the cells alone, without any stimulation. Allof the tests were carried out in triplicate. After incubation, the platewas emptied and subjected to 5 successive washings with PBS, 200 μl perwell. The cytokine-antibody complexes were stained by enzymatic reactionand the addition of a chromogenic substrate: 100 μl of secondaryantibody conjugated with alkaline phosphatase, distributed by Mabtech,diluted 1:200 in PBS 1× and 0.5% foetal bovine serum (FBS), wasdispensed into each individual well and, after incubation for 120′ atambient temperature, the substrate 100 μl (BCIP/NTB-plus), alsodistributed by Mabtech, was added. The reaction with the substrate wasstopped after 30-40′ by washing the plate with running water. Thenitrocellulose membrane was dried in air for at least 4 hours. Eachindividual spot which appeared on the base of the well corresponded tothe secretion of the cytokine of a single cell (spot forming cell: SFC).The spots were then counted automatically by means of an image-analysistool (AID ELISPOT Reader System, Amplimedical) controlled by softwarecapable of providing a quick and easy evaluation of the spots on thebasis of their size and intensity.

ELISPOT Method for Mucorales-Specific IL-4 Producing T Cells

96-well microtitration plates (Multiscreen HTS IP Sterile Plate),distributed by Millipore (Bedford, Mass., USA) and included in theELISPOT IL-10 kit distributed by Mabtech (Nacka Strand, Sweden) wereused for all of the ELISPOT tests. The bases of the wells of the platewere made of nitrocellulose and were coated with a specific IL-4monoclonal antibody. The plates were removed from the packaging andwashed 4 times with PBS 1× (200 μl/well). The plates were fixed with R10(200 μl/well) and incubated at ambient temperature for ≧30′.

The fixing means were removed and 100,000-250,000 cells in R10 weredispensed into the individual wells (final volume of 100-150 μl/well)and then stimulated with sonicated, germinated conidia, deactivated byheat (1−2×10⁵ conidia/ml) or with proteinaceous extract (6-10 μg/ml)during incubation at 37° C. in the presence of CO₂ (5%) for about 40hours. The positive controls were constituted by the same cellsincubated with phytohaemagglutinin (PHA) (25 μg/ml) or with a humananti-CD3 antibody (Mabtech) used at a dilution of 1:1,000; the negativecontrol was constituted by the cells alone, without any stimulation. Allof the tests were carried out in triplicate. After incubation, the platewas emptied and subjected to 5 successive washings with PBS, 200 μl perwell. The cytokine-antibody complexes were stained by enzymatic reactionand the addition of a chromogenic substrate: 100 μl of secondaryantibody conjugated with alkaline phosphatase, distributed by Mabtech,diluted 1:300 in PBS 1× and 0.5% foetal bovine serum (FBS), wasdispensed into each individual well and, after incubation for 120′ atambient temperature, the substrate, 100 μl (BCIP/NTB-plus), alsodistributed by Mabtech, was added. The reaction with the substrate wasstopped after 30-40′ by washing the plate with running water. Thenitrocellulose membrane was dried in air for at least 4 hours. Eachindividual spot which appeared on the base of the well corresponded tothe secretion of the cytokine of a single cell (spot forming cell: SFC).The spots were then counted automatically by means of an image-analysistool (AID ELISPOT Reader System, Amplimedical) controlled by softwarecapable of providing a quick and easy evaluation of the spots on thebasis of their size and intensity.

Preparation of the Antigen from Mucorales Conidia

After culture of Mucorales for three days on Saboraud agar medium, theconidia were collected by washing of the surface layer of the culturewith 30 ml of sterile water. The washing liquid was filtered throughsterile gauze and then centrifuged at 12,000 rpm for 10 minutes.

The supernatant liquid was removed and the conidia pellet thus obtainedwas resuspended in liquid Saboraud and stirred for 12 hours to achievegermination. The germinated conidia were washed in saline solution(PBS), deactivated by heat for 1 hour at 100° C., and counted. Theantigen (2-4 million germinated conidia) was then treated by sonicationwith the use of an ultrasound homogenizer reaching a maximum power of400 W and working at a frequency of 24 KHz. The conidia were subjectedto 4 sonication cycles, each of 10 seconds duration, at 70% of maximumpower. Finally, the germinated and sonicated conidia were resuspended inPBS or culture medium (1 ml) and were used immediately or stored at −20°C. and used subsequently.

The conidia thus obtained were used in the Elispot test at a finalconcentration of about 100,000-200,000 conidia/ml and in the CSA test ata concentration of 200,000-400,000 conidia/ml.

Preparation of the Antigen from Mucorales Hyphae

After culture of Mucorales on Saboraud agar medium, several cultureplates were scraped and the scraped product was suspended in 30 ml ofsterile water.

The suspension obtained was stirred in a vortex, filtered throughsterile gauze and centrifuged at 15,000 rpm for 10 minutes.

The supernatant liquid was then removed and the mycete pellet frozen for12 hours in liquid nitrogen.

The mycete pellet was then thawed and homogenized in a mortar with anequal volume of fungus/beads in 50 mM Tris-HCl buffer at a pH of about7.5.

The homogenate obtained was centrifuged at 14,000 for 20 minutes and theproteinaceous content was evaluated.

Finally, the hyphae obtained could be used directly in the ELISPOT testaccording to the present invention at a concentration of 6-10 μg/ml.Alternatively, the hyphae were frozen to −20° C. until the time of useaccording to the present invention.

Clinical Study

In order to evaluate whether the determination and the counting of theMucorales-specific IFN-γ-Th1, IL-10 (IL-10-Th2) and IL-4 (IL-4-Th2)producing T cells by means of the enzymatic immunospot (ELISPOT) andimmunofluorimetric (CSA) assays could improve the clinical diagnosis andmonitoring of IZ, clinical tests were carried out on a series of 13haematological patients with radiologically and clinically documentedneutropenia and infections and five healthy donors. Three of thehaematological patients with infections were suffering from provenInvasive Zygomycosis.

Mononucleated peripheral blood cells (PBMCs) were separated from eachpatient or healthy subject by centrifuging on a Ficoll-Hypaque gradient(Linaris, Bettingen am Main, Germany) and then put in culture, some in96-well polyvinylidene fluoride plates with anti-IFN-gamma, anti-IL-10and anti-IL-4 monoclonal antibodies (Mabtech, Nacka Strand, Sweden), andsome stimulated with the antigens described below in order then to beanalyzed with a cytofluorimeter. The cells were stimulated withgerminated conidia, deactivated by heat and sonicated, prepared fromMucorales isolated from the patient, and/or with a water-soluble cellextract of Mucorales with PHA and with anti-CD3. 1×10⁵ cells/well wereput in culture for sixteen hours in IFN-gamma assay whereas 3×10⁴cells/well and 2×10⁵ cells/well were put in culture for 40 hours inIL-10 and IL-4 assay, respectively.

All of the test conditions were implemented in triplicate and theresults were considered positive if: 1) the number of spot forming cells(SFC)/10⁶ cells in Mucorales-antigen-stimulated wells was twice as greatas in the control wells (cells in non-Mucorales-antigen-stimulatedwells) and there were at least 20 spots, and/or 2) the difference in SFCbetween negative and stimulated was greater than or equal to 5.

The first patient was a 54-year-old man who, on day 16 of thechemotherapy induction cycle for acute myeloid leukaemia (AML), duringthe neutropenic phase, had developed fever and a right pulmonary lesion.An empirical antibiotic treatment with glycopeptides and carbapenemesand treatment with L-amB at a dosage of 3 mg/kg/die were undertaken.Culture and molecular examinations of blood, urine, faeces andbronchioalveolar lavage liquid (BALf) were repeatedly negative for thepresence of bacteria, fungi or viruses. On day 22, a volumetricenlargement of the right pulmonary lesion was found. On day 36, afurther enlargement of the nodular consolidation area was observed. Onday 47, a surgical resection of the pulmonary lesion was performed. Onday 52, the histological report of the pulmonary biopsy was compatiblewith Invasive Mucormycosis. On day 58, cultural examination on thepulmonary biopsy and molecular examination characterized a mycete of thespecies Rizhopus pusillus. The patient achieved complete remission ofthe haemopathy and undertook secondary anti-mycotic prophylaxis withposaconazole and then underwent a consolidation chemotherapy cycle.

The second patient was a 68-year-old woman suffering from severeaplastic anaemia (SAA) who, on day 26 of immunosuppressive treatment,had fever and a voluminous para-tracheal lymphonodal conglomeration uponhigh-resolution computerized tomography (HRCT) of the thorax. Culturaland molecular examinations of blood, urine, and faeces were repeatedlynegative for the presence of bacteria, fungi or viruses. On day 28 ofthe immunosuppressive treatment, bronchoscopy showed infiltration of thetrachea with complete destruction of the cartilaginous structure.Multiple biopsies were performed. On day 33, histological examination ofthe tracheal biopsy showed fungal hyphae compatible with infection byfilamentous fungus, most probably Aspergillus. Treatment with L-amB wasundertaken at a dosage of 5 mg/kg/die. On day 35, cultural examinationon SAL liquid was positive for Mucor spp. On day 36 A Y-shaped trachealstent was put in place. On day 41, cultural examination performed on thetracheal biopsy was also positive for Mucor spp. A control HRCT of thethorax showed the tracheal compression unchanged. On day 49, anoesophageal ulcer by ab-extrinsec infiltration was found. On day 55, atracheo-oesophageal fistula was found. On day 64, posaconazole wascombined with the L-amB. On day 77, occlusion of the left branch of thetracheal stent by granulomatous reaction was recorded. On day 84, a newbronchoscopy was performed and it was impossible to remove the stent andopen the left bronchus. The patient's dyspnoea worsened progressivelyuntil it led to the patient's death on day 88 from the start ofimmunosuppressive therapy.

The third patient was a 36-year-old man suffering from acutepromyelocytic leukaemia (APL) who, on day 35 of induction treatment withanthracycline and all-transretinoic acid (ATRA) had developed diplopiaand cephalea. An encephalo-CT was negative. Morphological and culturalexaminations on cephalorachidian fluid were negative for the presence ofbacteria, fungi or viruses or for localization of APL. On day 42, owingto the appearance of sx palpebral ptoses, an encephalo-NMR was performedand neoformed tissue was found at the level of the sx cavernous sinus.On day 50, the appearance of sx eye pain was observed and, uponencephalo-CT, bone rarefaction of the sx sphenoidal sinus was found. Onday 60, a transphenoidal biopsy of the mucosa of the sx sphenoidal sinusand of the neoformed tissue of the cavernous sinus was performed. On day69, histological examination of the transcranial biopsy showedinflammatory tissue with non-septate fungal hyphae. The patient wassubjected to antifungal treatment with L-amB at a dosage of 5 mg/kg/die.On day 75, culture and molecular examination, also performed bymicromanipulation on an individual fungal cell, characterized InvasiveMucormycosis by Rizhopus spp. Posaconazole at a dosage of 800 mg/die wascombined with the treatment in progress. On day 82, the patient, who hadachieved complete remission of the haemopathy, was discharged withpurely oral treatment with posaconazole. Currently, the patient is stillin LAP molecular remission after having completed 4 maintenance cycleswith arsenic trioxide and ATRA. The treatment with posaconazole iscurrently still in progress. Upon a latest, recent check with NMR theinflammatory/infective tissue of the sx sphenoidal sinus had reduced insize and the symptomology, including the diplopia, was on the way tocomplete resolution.

In patient 1, the ELISPOT method according to the present invention wasperformed at the time at which chemotherapy started (day 1), between thesecond and third HRCTs (day 27), 2 days before the third HRCT of thethorax (day 34) and subsequently on days 51 and 64 and on day 238. TheELISPOT was negative for the presence of Mucorales-specific T cells inthe first and last determinations, in the absence and upon resolution ofthe infective pathology, whereas it was positive in the determinationsthat were performed in concomitance with the pulmonary infection byMucorales. In particular, the ELISPOT method according to the presentinvention showed: 1) the presence of Mucorales-specific T cells,polarized towards a Th2-type immunity and producing IL-10 on days +27and +64; 2) the presence of Mucorales-specific T cells polarized towardsa Th1-type immunity and producing IFN-γ in the determinations of days+27, +34 and +51; 3) a decreasing ratio between the number of IL-10producing cells and that of IFN-γ producing cells in the determinationsof days +27, +34 and +51 (FIG. 1A). In patient 2, the ELISPOT methodaccording to the present invention was performed on peripheral bloodsamples collected on days 36, 55, 68, 75 and 82 from the start of theimmunosuppressive therapy, respectively. The ELISPOT was positive forthe presence of solely Interleukin-10 producing Mucorales-specific Tcells upon the first determination (36) and positive for the presence ofboth IL-4 and IL-10 producing Mucorales-specific T cells, and henceindicative of a Th2-type response, in the subsequent determinations (55,68, 75, 82). In the three determinations (55, 68, 72), the ELISPOT waspositive for the presence of Mucorales-specific interferon-gamma (IFN-γ)producing T cells which are indicative of a Th1-type response (FIG. 1B).

In patient 3, the ELISPOT method was performed on day 75 and on day 95of the induction therapy, on day 48 (day 137 from the start of thetreatment), on day 55 (day 143 from the start of treatment) of themaintenance therapy, and on day 217 from the start of the treatment. TheELISPOT was positive solely for a Mucorales-specific, IL-10 producing Tcell response in the first and second determinations and for aMucorales-specific IFN-γ producing and hence Th1 and IL-4 producing Tcell response in the third and fourth determinations. The ELISPOT wasnegative for Mucorales-specific T cells in the last determination inconcomitance with the almost complete resolution of the infectiveepisode (FIG. 1C).

Clinical Results

The method according to the present invention thus provided proof ofMucormycosis in all three patients. In particular, the positivity of theELISPOT according to the present invention was the sole proof of theinfection in patient 1, as early as day 27. In this patient, thediagnosis was reached, one month afterwards, solely by surgicalintervention, only on day 42, all of the other methods having beennegative. In patient 2 and in patient 3, ELISPOT provided a validsupport for histological and cultural diagnosis, representing the solenon-cultural and non-invasive positive result.

FIG. 2 shows the results of ELISPOTs in patients affected by pulmonitiswith non-fungal aetiology, used as negative controls.

1. A method for the diagnosis and/or monitoring of Mucormycosiscomprising the execution of an in vitro assay in which the biologicalfluid taken from the patient is put in contact with an antigen ofMucorales, and subsequent checking for the presence of specific IFN-γ,producing T cells, specific IL-10 producing T cells and/or specific IL-4producing T cells.
 2. A method according to claim 1, characterized inthat the Mucormycosis is Invasive Mucormycosis.
 3. A method according toclaim 1, characterized in that the assay is an immunoenzymatic assay. 4.A method according to claim 3, characterized in that the immunoenzymaticassay is the ELISPOT assay.
 5. A method according to claim 3,characterized in that the immunoenzymatic assay is the Quantiferonassay.
 6. A method according to claim 1, characterized in that the assayis an immunocytofluorimetric assay.
 7. A method according to claim 6,characterized in that the immunocytofluorimetric assay is selected fromCSA assay and ICS assay.
 8. A method according to claim 1, characterizedin that the Mucorales antigen is an extractable antigen.
 9. A methodaccording to claim 1, characterized in that the extractable antigen isobtained from Mucorales conidia and/or hyphae.
 10. A method according toclaim 9, characterized in that it comprises the following steps:incubation of the conidia for 2-4 days, subsequent collection of theconidia by washing of the fungal culture with sterile water, subsequentfiltration and centrifuging of the washing liquid, subsequent removal ofthe supernatant liquid, subsequent suspension in liquid Saboraud andgermination with stirring, preferably for 6-18 hours, subsequent washingin saline solution, subsequent boiling and centrifuging, subsequentsonication of the germinated conidia de-activated by heat, subsequentsuspension of culture medium in saline solution, and subsequent optionalfreezing.
 11. A method according to claim 9, characterized in that itcomprises the following steps: collection of hyphae from culture plates,preferably by scraping, subsequent suspension in sterile water,subsequent filtration and centrifuging, subsequent removal of thesupernatant liquid, subsequent freezing and thawing of the mycetepellet, subsequent homogenization of the mycete in the presence of avolume of beads made of inert material, preferably glass, approximatelyequal to the volume of the mycete, subsequent centrifuging, subsequentoptional freezing.
 12. A method according to claim 1, characterized inthat the biological fluid is selected from blood, bronchioalveolarlavage liquid and/or pleural liquid.
 13. A method according to claim 1,characterized in that the positivity threshold for the specific IFN-γproducing T cells, the specific IL-10 producing T cells, and/or thespecific IL-4 producing T cells is between 2 and 10 SFCs, preferably 5SFCs.
 14. A method according to claim 1, characterized in that theantigen is a recombinant protein of Mucorales and/or at least onepeptide of Mucorales.